Isolation and Antimicrobial Resistance Patterns of Bacterial Pathogens from Community-Acquired Pneumonia at Adama Hospital Medical College, Adama, Ethiopia

Community-acquired pneumonia (CAP) is a major cause of morbidity and mortality worldwide. It also contributes significantly to hospital admissions, particularly in low-income countries such as Ethiopia, where it accounts for major public health problems. This could be attributed to the increasing prevalence of antibiotic-resistant pathogens in CAP patients. This study aimed to identify and assess the antibiotic resistance patterns of bacterial isolates from CAP patients at the Adama Hospital Medical College in Adama City, Ethiopia. A cross-sectional study was conducted from November 10, 2022, to November 30, 2023. Demographic, clinical data, and sputum samples were collected from patients with CAP (n = 369). Sputum samples were subjected to standard microbiological procedures, including culture, Gram staining, and a panel of different biochemical tests for the identification of pathogenic bacterial isolates. The Kirby–Bauer disc diffusion method was used for drug susceptibility testing. Descriptive statistics were computed by using SPSS (version 26). Of the 369 patients with CAP, bacterial pathogens were identified in 31.7% (n = 117, 95% CI: 27.0%–36.7%). The most common isolates were Moraxella catarrhalis (n = 15; 12.8%), Staphylococcus aureus (n = 15; 12.8%), Klebsiella pneumoniae (n = 12; 10.3%), Escherichia coli (n = 11; 9.4%), Pseudomonas aeruginosa (n = 11; 9.4%), Enterobacter species (n = 11; 9.4%), and Citrobacter species (n = 11; 9.4%). Among the identified isolates, resistance rates were high in Enterobacteriaceae, followed by Gram-positive bacteria, and non-Enterobacteriaceae. Overall, 68 (58.1%) of the identified bacterial isolates were multidrug resistant (MDR), with K. pneumoniae accounting for the highest proportion of multidrug resistant isolates (91.7%), while P. aeruginosa accounted for the lowest proportion (9.1%) of MDR isolates. This study revealed a high prevalence (31.7%) of bacterial pathogens in CAP patients and higher (58.1%) MDR bacterial pathogens. Therefore, regular surveillance and monitoring systems are warranted for assessing predominant pathogens and antibiotic resistance patterns.


Introduction
Community-acquired pneumonia is a common cause of morbidity and mortality worldwide, with a signifcant impact on the healthcare system [1,2].Te estimated worldwide incidence of CAP varies from 1.5 to 14 cases per 1000 person-years [3,4].It is also prevalent in developing countries, causing approximately 700,000 deaths annually [5].In sub-Saharan Africa, the burden of CAP is increasing, and it is among the major causes of morbidity and mortality among adults [6].In Ethiopia, although the epidemiology of CAP varies from place to place, a study conducted in the Tigray region revealed a prevalence of 16% in adults [7].Te recent systematic review on the prevalence of CAP in Ethiopia showed that the disease is the second leading cause of death in the country [8].
Community-acquired pneumonia is usually diagnosed by employing a combination of clinical and radiological techniques, and empirical treatment is commonly employed, especially in developing countries [9].However, it is crucial to understand the specifc causes of pneumonia to make informed decisions regarding antibiotic treatment.Tis is due to variations in underlying causes that can lead to an inadequate response to therapy.Studies conducted in various regions globally have identifed bacterial pathogens as common microorganisms responsible for causing CAP [10][11][12].Te common bacterial pathogens responsible for CAP include S. pneumoniae, H. infuenzae, E. coli, S. aureus, M. catarrhalis, P. aeruginosa, and other Gram-negative bacilli [10,13].However, the etiology of CAP has undergone a substantial evolution since the preantibiotic era.Tese etiological variations are due to the irrational use of antibiotics, environmental pollution, advancements in diagnostic techniques and tools, increased awareness of the disease, and widespread introduction of pneumococcal conjugate vaccines [6,10].Although S. pneumoniae remains the most commonly reported bacterial etiology of CAP worldwide [14], there are reports of an increasing incidence of bacteria such as M. catarrhalis [15,16] and P. aeruginosa [17], particularly in immunocompromised patients.
In addition to accurately identifying the causes of CAP, profling drug resistance of the identifed bacterial pathogens plays a crucial role in efectively treating patients.Te extensive utilization and misuse of antibiotics have led to a global surge in antibiotic resistance, contributing to the emergence of drug-resistant pathogens [18,19].Te widespread prevalence of antibiotic resistance and dissemination of MDR strains are major concerns and present signifcant challenges in the healthcare feld [20].Te spread of MDR bacteria within the community has become a critical public health threat, posing considerable difculties for clinicians owing to high mortality rates and limited treatment options [21].Studies conducted in some regions of Ethiopia have revealed a high prevalence of mono-and multidrug-resistant bacterial pathogens among patients with CAP [22,23].Tese fndings highlight the signifcance of conducting similar studies in diferent settings to provide evidence-based interventions for efectively controlling the spread of drugresistant pathogens.
Terefore, studying the etiological pathogens of CAP and their antibiotic resistance patterns is important to reduce the morbidity and mortality associated with community acquired pneumonia.Tis study aimed to identify the etiologies of CAP and their antibiotic resistance patterns at Adama Hospital Medical College (AHMC) in Adama City, Ethiopia.

Study Area and Period.
Te study was conducted at AHMC, Adama, Ethiopia, which is situated in the Oromia region 100 km Southeast of Addis Ababa.AHMC is a referral hospital in Adama and has an estimated catchment of six million individuals from fve regions: Oromia, Amhara, Afar, Somali, and Dire-Dawa.With a capacity of 527 beds, the hospital admits and treats an average of 1000 patients daily across 15 outpatient departments, spanning 10 different specialties.Te study was conducted from November 10, 2022, to November 30, 2023.

Population and Sample.
A total of 369 adults aged ≥18 years who were clinically suspected to have CAP consented to participate in the study.Patients with severe immunosuppression, cough lasting for more than 2 weeks, COVID-19 infection, pregnancy, children under 18 years of age, recent antibiotic use within the past 2 weeks, individuals at risk of hospital-acquired pneumonia, and critically ill patients unable to expectorate sputum were excluded from the study.

Data Collection and Laboratory Methods.
Sociodemographic characteristics (age, sex, marital status, family size, educational level, occupation, and monthly income) and pneumonia risk factors such as smoking and travel history, current or past comorbidities, alcohol habits, and prior antibiotic treatment information were collected from patients diagnosed with CAP, presenting with pneumonia-like symptoms or confrmed by X-ray.Additionally, clinical history, physical examination, and laboratory assessment, including initial signs and symptoms of CAP, chest radiograph fndings, blood pressure, respiratory rate, pulse rate, oxygen saturation, and vaccination history were recorded.Sputum specimens were obtained from adult patients with CAP by using a sputum cup.Patients were instructed to take deep breaths and then cough deeply and vigorously to produce a minimum of 2 mL of sputum.Te sputum cup containing the specimen was appropriately labeled with a unique sample number and immediately transported to the Adama Public Health Research and Referral Laboratory Center for microbiological analysis.

Bacterial Isolation.
Isolation and identifcation of pathogenic bacteria were conducted using conventional microbiological techniques as described in the Clinical Laboratory Standards Institute (CLSI) guidelines [24].Briefy, a purulent portion of the sputum specimen was inoculated on a Blood Agar Plate (BAP), Chocolate Agar Plate (CAP), and MacConkey Agar Plate (MAP) using a sterile loop.Te inoculated CAP and BAP were incubated in a candle jar under 5-10% CO 2 at 37 °C for 24-48 hours.Te inoculated MAP was aerobically incubated for 24 h at 37 °C.Positive growth on BAP and MAP was subcultured on BAP to identify pure colonies.Te pure bacterial isolates were characterized based on colony morphology, hemolysis, Gram staining, and a panel of biochemical tests.Grampositive bacteria were identifed using catalase, coagulase/mannitol salt agar (MSA), optochin, and bile solubility tests, while Gram-negative isolates were identifed using the oxidase test, (H 2 S, indole, and motility tests (SIM)), urea test, triple sugar iron agar (TSI), citrate utilization tests, and lysine iron agar (LIA) test.Pure colonies were stored in 20% glycerol at −80 °C.

Quality Control.
All standard operating procedures (SOPs), including staining reagents, culture media preparation, biochemical tests, and antibiotic susceptibility tests, were strictly followed.Te sterility of the prepared culture media was checked by incubating 5% of the batch at 37 °C overnight and evaluated for possible bacterial growth.Quality control of all prepared media was also checked by inoculating standard strains such as E. coli (ATCC 25922), S. aureus (ATCC 25923), S. pneumoniae (ATCC 49619), P. aeruginosa (ATCC 27853), P. mirabilis (ATCC 35659), K. pneumonia (ATCCC 700603), A. baumanni (ATCC 19606), and H. infuenzae (ATCC 49766).Furthermore, the potency of the tested antibiotics was evaluated using the control strains.McFarland standard (0.5 McFarland) was used to standardize the inoculum density of the bacterial suspension.

Data Analysis and Interpretation.
Te data were checked for completeness, coded, and entered into a Microsoft Excel spreadsheet.Subsequently, the data were imported into SPSS version 26 (IBM-SPSS Inc., Chicago, IL, US), coded, and analyzed using descriptive statistics and 95% CI for prevalence.

Sociodemographic, Clinical, Behavioral Characteristics, and Hospital Outcome of the Study Participants.
Of the 369 enrolled patients, 199 (53.9%) were males.Te ages of the participants ranged from 18 to 87 years, with a mean (±standard deviation [SD]) of 40.41 [±17.14].Most of the study participants, 180 (48.8%), were within the age range of 18-35 years.Te majority of the participants, 281 (76.2%), were married, and 156 (42.3%) participants were living in a family size of 3-5 members per household.123 (33.3%) of the participants had no formal education, and more than half 189 (51.2%) of the participants had no monthly income (Table 1).Among these patients, 296 (80.2%) were hospitalized and 73 (19.8%) were treated as outpatients.
A total of 359 (97.3%) patients had cough, 246 (66.7%) reported fatigue, and 60 (16.3%) had a history of prior antibiotic intake in the previous 3 months.No single patient had received the pneumococcal vaccine.Supplementary Table 1 summarizes the most common clinical, behavioral, and comorbidities of patients with CAP.

Journal of Tropical Medicine
S. pneumonia ( C. diphtheria (

Discussion
In the present study, bacterial isolates were identifed in 31.7% of the CAP patients, which is lower than the fndings reported elsewhere in Ethiopia: Dessie town, Northeastern Ethiopia, 46.3% [26]; Gondar, Northwestern Ethiopia, 39.4% [27]; and Jimma town, Southwestern Ethiopia, 50.0%[28].One possible reason for the lower level of bacterial isolates compared to the previous studies conducted in Ethiopia may be attributed to the widespread usage of antibiotics before presentation to the hospital, as 16.3% of the participants in this study had a history of prior antibiotic intake.On the other hand, the other possible explanation for the lower level of bacterial isolation from CAP patients in our study could also be associated with other causative agents, such as viral causes, which could not be identifed by the diagnostic approaches we used in this study.In support of this, study by Jennings et al., [29] and systematic review by Burk et al. [30] showed that viral pathogens could be the etiology of community-acquired pneumonia in 29% and 24.5% of the cases.
Our fndings showed a higher prevalence of Gramnegative bacteria compared to Gram-positive bacteria, which is consistent with previous studies conducted in Ethiopia [27,31,32].Te changing trend in the etiology of CAP from predominantly Gram-positive bacteria, such as S. pneumoniae [14], to Gram-negative bacteria (GNB) could be attributed to several factors.Tese factors include limited vaccine coverage targeting GNB, inadequate antimicrobial stewardship leading to the irrational use of drugs, and the emergence and spread of drug resistance among GNB [13].Te shift in the trend may also be associated with the production of extended-spectrum β-lactamases (ESBL) by Gram-negative bacteria, as the prevalence of ESBLproducing isolates increases, resulting in antimicrobial resistance to drugs [33].
Te most frequent isolates in the current study were M. catarrhalis and S. aureus.Tis fnding is consistent with studies conducted in Uganda, where Moraxella species were found to be more prevalent [34] and retrospective observational cohort study conducted in Vienna, Austria [35], where S. aureus was the predominant isolate.However, the fndings of this study was in variation with the fndings conducted in Jimma town, Southwestern Ethiopia [28] where K. pneumonia and P. aeruginosa were the most frequent isolates followed by other species.Te predominant M. catarrhalis in this fnding showed the signifcant emergency of this pathogen as an etiology of pneumonia in recent years [36].Te prevalence of S. aureus in CAP has also increased in recent decades, with high emergence of new lineages of MRSA in the community [37].
Drug resistance is a signifcant public health challenge, with the increasing emergence and spread of drug-resistant pathogens.Our study highlights this issue, as a majority of the identifed bacterial pathogens demonstrated resistance to currently available drugs.Among the identifed bacterial isolates, Enterobacteriaceae exhibited high levels of resistance to ampicillin and cephalosporins, consistent with a study conducted at four diferent hospitals in Ethiopia [38].Te increasing trend of drug resistance in Enterobacteriaceae is a common observation in Ethiopia [39,40], likely attributable to the prevalent misuse of drugs without proper prescription by physicians.Our study also showed that K. pneumoniae isolates displayed complete resistance to ampicillin, cefepime, and cefazolin, as well as high resistance to ceftriaxone, ceftazidime, cefuroxime, and trimethoprim-sulfamethoxazole.Our fndings are in line with the Ethiopian annual antimicrobial surveillance report, which indicated a high resistance of K. pneumoniae isolates to cephalosporins and trimethoprim-sulfamethoxazole [41].M. catarrhalis showed 73.3%, 60.0%, and 53.3% resistance rates to azithromycin, Journal of Tropical Medicine erythromycin, and trimethoprim-sulfamethoxazole, whereas P. aeruginosa showed 45.5% resistance rate to cefepime.In contrary with our fndings, a study done in China showed a high level of sensitivity of M. catarrhalis to azithromycin, 100%, and trimethoprim-sulfamethoxazole, 67.7% [11].Te higher resistance pattern observed in this study could be associated with several factors, including the widespread irrational use of drugs prior to patients visiting health facilities and the increasing circulation of drug-resistant isolates in the community warranting continues monitoring of the resistance profle in the community.Nowadays, bacterial resistance to multiple drugs is becoming a common phenomenon, and our study also supports this fact.In our study, 58.1% of the identifed bacterial isolates were found to be MDR.Tis prevalence is higher compared to a study conducted in Hawassa University Comprehensive Specialized Hospital (HUCSH), Southern Ethiopia [22], which reported an MDR rate of 32.4%.However, the MDR rate in our study is lower than that reported in previous studies conducted in Gondar, Northwestern Ethiopia [27], Felege Hiwot Referral Hospital, Northwestern Ethiopia [23], and Dessie town, Northeastern Ethiopia [26], which reported overall MDR rates of 72.2%, 76.0%, and 84.6%, respectively.One possible explanation for this diference could be geographical variations in bacterial CAP among adults.It is well known that resistance patterns of bacteria can vary geographically owing to factors such as local antimicrobial usage, healthcare practices, and population characteristics.Tese variations can contribute to the observed diferences in the rates of MDR among diferent bacterial families in diferent regions or settings.
Our study also revealed that MDR was observed in 75.8% of Enterobacteriaceae isolates, 47.8% of Gram-positive bacteria isolates, and 21.4% of non-Enterobacteriaceae isolates.Tese fndings are comparable to a study conducted at four selected hospitals in central, southern, and northern Ethiopia [38], where Enterobacteriaceae had a higher MDR rate of 83.2%, followed by Gram-positive bacteria (47.8%) and non-Enterobacteriaceae (47.5%).Te highest MDR isolates were observed in K. pneumonia (91.7%).Similar to our fnding, high MDR K. pneumonia isolates were also reported from four selected hospitals in central, southern, and northern Ethiopia [38], Tikur Anbessa Specialized Hospital (TASH), Addis Ababa, Ethiopia [41], and Felege Hiwot Referral Hospital, Northwestern Ethiopia [23], with 95.5%, 98.5%, and 100% MDR rates, respectively.Based on our fndings, we can speculate that MDR isolates, particularly MDR K. pneumoniae isolates, which cannot be treated by commonly used drugs, are widely distributed in various settings in Ethiopia, requiring a concerted efort to control and prevent their spread.

Conclusions
Tis study revealed a high prevalence of bacterial pathogens in CAP patients.Te antibiotic resistance profle observed in the study area was also found to be alarming.Terefore, it is crucial to strengthen actions aimed at reducing the impact of antimicrobial resistance.

Table 2 :
Antimicrobial resistance and susceptibility patterns of Enterobacteriaceae isolated from CAP patients, AHMC, 2023 (n �

Table 3 :
Antimicrobial resistance and susceptibility pattern of non-Enterobacteriaceae bacteria isolated from CAP patients, AHMC, 2023 (n �